TEM TOMOGRAPHY OF A DROSOPHILA ATP6 MUTATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要 我们对导致寿命缩短和神经退化的突变的研究使我们发现了线粒体基因ATP6(ATPA的同源基因)中的一种母系遗传突变。该基因编码F1F0三磷酸腺苷合成酶的H+通道成分，通过旋转催化将离子梯度耗散与三磷酸腺苷的产生结合起来。在人类中，当突变接近同型时，ATP6的损害通常表现在疾病中。例如，携带线粒体ATP6基因(L156R)最具特征的突变的人患有MILS、NARP，或者是正常的，这取决于突变的异质性(分别为~85%、~70%或~70%突变)。在我们的突变体ATP6[1]中，突变几乎是同型的(98.4%是突变体)。电子显微镜分析显示ATP6[1]突变体的线粒体中有异常的冠状突起(图1)。我们用体视学方法获得了显微照片，并发现ATP6[1]的线粒体密度(31？3/100mm2)与野生型(35？2/100mm2，p=0.4)没有显著差异。我们希望更好地了解这种形态异常，以补充我们正在进行的这些线粒体的功能研究。具体地说，内部线粒体膜是囊状的(如二维电子显微镜所示)还是管状的？内线粒体膜与外线粒体膜是否保持分离？5纳米分辨率的电子显微镜断层扫描非常适合直接解决这两个问题，这两个问题对线粒体的功能有深远的影响。我们的2D电子显微镜的质量和我们在突变体中看到异常线粒体的高频表明，拟议的项目将不会过于繁重，并将产生有启发性的结果。 图1：老化大脑的透射电子显微镜分析显示ATP6[1]突变体的线粒体形态异常(B)。N&gt；3个基因型。定量显示异常线粒体的频率很高(占总线粒体的60%，C)。与野生型比较(P&lt；0.01，t检验)。 在以前的报告所述期间，收到了来自野生型和ATP6突变体的阻断。由两个人组成的双倾斜重建。伦肯博士使用Tecnai F20从突变体身上收集了额外的单倾斜重建。使用跟踪和阈值技术制作了表面渲染模型。在突变体的线粒体中，似乎有一个从外周管到中央小泡的径向进展。改进了表面模型，制作了动画电影。这篇论文发表时，杂志封面上使用了其中一个曲面模型： +Celotto，A.M.，Frank，A.C.，McGrath，S.W.，Fersterad，T.，Van Voorhies，W.A.Buttle，K.F.，Mannella，C.A.，Palladino，M.J.(2006)果蝇线粒体脑肌病。J.神经学26：810-820。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT Our studies of mutations that cause shortened lifespan and neurodegeneration have led us to identify a maternally inherited mutation in the mitochondrial gene ATP6 (ortholog of ATPa). This gene encodes the H+ channel component of the F1F0 ATP synthase, which couples ion gradient dissipation with ATP production via rotary catalysis. In humans, impairment of ATP6 generally manifests in diseases when the mutation is nearly homoplastic. For example, humans bearing the best-characterized mutation in the mitochondrial ATP6 gene (L156R) have MILS, NARP, or are normal depending upon the heteroplasmy of the mutation (>~85%, ~70-85% or <~70% mutant, respectively). In our mutant, ATP6[1] the mutation in nearly homoplastic (98¿4 % mutant). TEM analysis revealed aberrant cristae in mitochondria from ATP6[1] mutants (Figure 1). We have acquired the micrographs using stereological protocols and have discovered the density of mitochondria in ATP6[1] (31 ¿ 3 per 100mM2) does not appear to be significantly different than wild type (35 ¿ 2 per 100mM2, p=0.4). We would like to better understand this morphological aberration to complement our ongoing functional studies of these mitochondria. Specifically, is the internal mitochondiral membrane vesiciular (as it appears in 2D TEM) or is it tubular? Does the internal membrane remain separate from the outer mitochondrial membrane? TEM tomography with a 5nm resolution is extremely well-suited to directly address both of these questions, which have profound consequences on the function of mitochondria. The quality of our 2D TEM and the high frequency with which we see aberrant mitochondria in our mutants suggests the proposed project will not be overly burdensome and will yield instructive results. Figure 1: TEM analysis of aged brains reveals aberrant mitochondrial morphology in ATP6[1] mutants (B). N > 3 each genotype. Quantification demonstrates a high frequency of abnormal mitochondria (>60% of total mitochondria, C). Comparison to wild type (* p<0.01, T-test). In the previous reporting periods, blocks from wild-type and ATP6 mutant were received. A double-tilt reconstruction made from each. Dr. Renken collected an additional single-tilt reconstruction from the mutant, using the Tecnai F20. Surface-rendered models, using both tracing and thresholding techniques, were made. In mitochondria from the mutant, there appears to be a radial progression from peripheral tubes to central vesicles. Surface models were improved, and animation movies were made. One of the surface models was used on the journal cover when the paper was published: + Celotto, A.M., Frank, A.C., McGrath, S.W., Fergestad, T., Van Voorhies, W.A. Buttle, K.F., Mannella, C.A., Palladino, M.J. (2006) Mitochondrial encephalomyopathy in Drosophila. J. Neurosci. 26:810-820.